Snakemake implementation of VirusSeeker Virome workflow.

Setup environment and install prerequisites

Install miniconda

Download and install miniconda https://conda.io/docs/user-guide/install/index.html. In case of Linux, following should work:

wget https://repo.continuum.io/miniconda/Miniconda3-latest-Linux-x86_64.sh
bash Miniconda3-latest-Linux-x86_64.sh

Install environment

Create conda environment with snakemake. There are two options:

1. If you want to upload your results to Zenodo, then you need snakemake Zenodo remote provider, which is currently implemented in zenodo-simple branch in my forked snakemake repo.

First, clone snakemake repo and checkout zenodo-simple branch:

git clone https://[email protected]/tpall/snakemake.git
cd snakemake
git checkout zenodo-simple

Then, create conda environment, install prerequisites and snakemake:

conda env create -f environment.yml -n snakemake
source activate snakemake
pip install -e .

2. Alternatively, if you don't want to upload your results to Zenodo, you can create conda environment and install snakemake 'normally':

conda create -n snakemake -c bioconda -c conda-forge snakemake
source activate snakemake

Setup databases

Together all databases will occupy ~250GB+ from your HD.

BLAST databases

1. Download BLAST version 5 databases

Download version 5 BLAST databases using these instructions https://ftp.ncbi.nlm.nih.gov/blast/db/v5/blastdbv5.pdf

Briefly, you can use update_blastdb.pl script from BLAST+ software bundle to update/download BLAST databases.

To get BLAST, you can start by creating conda environment with blast+ like so:

conda env create -n blastenv
conda blastenv activate
conda install -c bioconda blast

Change working directory to location where you want BLAST databases to be installed, e.g. $HOME/databases/blast.

mkdir -p $HOME/databases/blast
cd $HOME/databases/blast

Use update_blastdb.pl (included with the BLAST+ package) to check available version 5 databases, use the --blastdb_version flag:

update_blastdb.pl --blastdb_version 5 --showall

Download nt_v5 and nr_v5 databases (takes time and might need restarting if connection drops):

update_blastdb.pl --blastdb_version 5 nt_v5 --decompress
update_blastdb.pl --blastdb_version 5 nr_v5 --decompress

2. Setup BLASTDB environment variable Edit $HOME/.bashrc file to permanently add BLASTDB variable to your shell environment

echo 'export BLASTDB=$HOME/databases/blast' >> $HOME/.bashrc
source $HOME/.bashrc
echo $BLASTDB

Download reference genome databases

1. Human reference genome.

First, create a directory for the reference genome sequence file, e.g mkdir -p $HOME/databases/ref_genomes && cd $HOME/databases/ref_genomes.

Then, human refgenome human_g1k_v37.fasta.gz sequences file can be obtained like so:

wget --continue ftp://ftp.ncbi.nlm.nih.gov/1000genomes/ftp/technical/reference/human_g1k_v37.fasta.gz

2. Bacterial reference genome sequences.

Create a directory for the bacteria reference sequence files. Download all *genomic.fna.gz files to the directory by using command.

wget --recursive --continue ftp://ftp.ncbi.nlm.nih.gov/refseq/release/bacteria/*genomic.fna.gz

Unzip the files and concatenate all the files into a single file. Use "bwa index" command to create index for the BWA algorithm.

3. Add paths to human_g1k_v37.fasta and Bacteria_ref_genome.fna to environment variables.

echo 'export REF_GENOME_HUMAN=$HOME/databases/ref_genomes/human_g1k_v37.fasta' >> $HOME/.bashrc
echo 'export REF_BACTERIA=$HOME/databases/bacteria_ref_sequence/Bacteria_ref_genome.fna' >> $HOME/.bashrc
source $HOME/.bashrc

Install workflow

Clone this repo and cd to repo (Change URL accordingly if using HTTPS)

git clone [email protected]:avilab/quantify-virome.git
cd quantify-virome

Example

Dry run

snakemake -n

Create workflow graph

snakemake -d .test --dag | dot -Tsvg > graph/dag.svg

Run workflow

This workflow is designed to be run in cluster. cluster.json configuration file may need some customisation, for example partition name. Memory nad maximum runtime limits are optimised for 100 splits. Number of splits can be specified in config.yaml file with n_files option (currently n_files is 2). Installation of software dependencies is taken care by conda, hence there is software installation overhead when you run this workflow for the first time in new environment.

Example workflow submission script for slurm cluster, where values for job name, cluster partition name, time and memory constraints, and slurm log path (output) are taken from cluster.json:

snakemake -j --use-conda --cluster-config cluster.json  \
             --cluster "sbatch -J {cluster.name} \
             -p {cluster.partition} \
             -t {cluster.time} \
             --mem {cluster.mem} \
             --output {cluster.output}"

You may want to use also following flags when running this workflow in cluster:

--max-jobs-per-second 1 --max-status-checks-per-second 10 --rerun-incomplete --keep-going

All other possible snakemake execution options can be printed by calling snakemake -h.

Exit/deactivate environment

Conda environment can be closed with the following command when work is finished:

source deactivate

Workflow graph

For technical reasons, workflow is split into two parts, virome and taxonomy, that can be run separately, but taxonomy depends on the output of virome. Virome subworkflow (virome.snakefile) munges, masks, and blasts input sequences. Taxonomy subworkflow (Snakefile) merges blast results with taxonomy data and generates report.

Figure 1. Virome workflow graph with test sample split into two (default = 20) subfiles for parallel processing. Outputs parsed BLAST results.